STALARD: Selective Target Amplification for Low-Abundance RNA Detection
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Background
Accurate quantification of RNA isoforms is critical for understanding gene regulation. However, conventional reverse transcription-quantitative real-time PCR (RT-qPCR) has limited sensitivity for low-abundance transcript isoforms, as quantification cycle (Cq) values above 30 are often considered unreliable. While transcriptome-wide analyses can address this limitation, they require costly deep sequencing and complex bioinformatics. Moreover, isoform-specific qPCR is often confounded by differential primer efficiency when comparing similar transcripts.
Results
To overcome the sensitivity and amplification bias limitations of conventional RT-qPCR for detecting known low-abundance and alternatively spliced transcripts, we developed STALARD ( Selective Target Amplification for Low-Abundance RNA Detection ), a rapid (< 2 h) and targeted two-step RT-PCR method using standard laboratory reagents. STALARD selectively amplifies polyadenylated transcripts sharing a known 5′-end sequence, enabling efficient quantification of low-abundance isoforms. When applied to Arabidopsis thaliana , STALARD successfully amplified the low-abundance VIN3 transcript to reliably quantifiable levels. Amplification of FLM , MAF2 , EIN4 , and ATX2 isoforms by STALARD reflected known splicing changes during vernalization, including cases where conventional RT-qPCR failed to detect relevant isoforms. STALARD also enabled consistent quantification of the extremely low-abundance antisense transcript COOLAIR , resolving inconsistencies reported in previous studies. In combination with nanopore sequencing, STALARD further revealed novel COOLAIR polyadenylation sites not captured by existing annotations.
Conclusion
STALARD provides a sensitive, simple, and accessible method for isoform-level quantification of low-abundance transcripts that share a known 5’-end sequence. Its compatibility with both qPCR and long-read sequencing makes it a versatile tool for analyzing transcript variants and identifying previously uncharacterized 3′-end structures, provided that isoform-specific 5′-end sequences are known in advance.
